The ripe fruit of Capsicum species are a well known, important source of a variety of carotenoids, including two main groups: carotenes and oxygenated carotene derivatives, commonly referred to as xanthophylls. For example, Capsicum species can contain capsanthin, capsorubin, cryptoxanthin, zeaxanthin, lutein, and other carotenoids that have substantial nutritional and medicinal value. Carotenoids are yellow, red and orange pigments which are widely distributed in nature. The carotenes refer to those carotenoids which only contain carbon and hydrogen atoms; examples include alpha and beta carotene and lycopene. Xanthophylls refer to carotenoids that contain one or more oxygen atoms, such as zeaxanthin, lutein, capsanthin, capsorubin, astaxanthin, cryptoxanthin, violaxanthin, and antheraxanthin. The hydroxyl-containing carotenoids are often found as esters in the plant material. They usually are found as diesters of fatty acids containing from eight to twenty carbon atoms. Examples of these fatty acids include linoleic, palmitic, oleic, linolenic, myristic, stearic, lauric, and the like.
Epidemiological studies have shown that frequent and regular consumption of carotenoids reduces risks of chronic disorders, such as cardiovascular diseases [Kohlmeier et al., (1995)] or cancer [Murakoshi et al., (1992); Levy et al. (1995); Tanaka et al., 1994); Ito et al. (2005), Connor et al. (2004), and Rock et al. (2005)]. Carotenoids may also function as antioxidants in disease prevention. Carotenoids have been found to be membrane antioxidants perhaps due to their reactivity with singlet oxygen and oxygen free radicals. Singlet oxygen has been shown to be capable of damaging proteins, lipids and DNA in biological systems. The potential cancer chemopreventive activity of carotenoids may be attributable to their antioxidant activity. Free radials generated in the body during metabolism can damage eye tissue. Eye tissue contains polyunsaturated fatty acids, which are susceptible to damage by free radicals and oxidative stress. In eye tissues, antioxidants such as zeaxanthin and lutein help prevent this damage.
Lutein and zeaxanthin are highly concentrated in the macula of the eye, with zeaxanthin being most highly concentrated at the center of the macula. The macula is a small area of the retina responsible for central vision and high visual acuity. These two xanthophylls protect the macula from the damaging photo-oxidative effects of short-wave UV radiation.
Xanthophylls such as zeaxanthin, lutein and cryptoxanthin have been shown to reduce the risk of age related macular degeneration (Moeller, et al., 2000 and Seddon, 1994), demonstrate control over LDL cholesterol (Chopra, et al., 1994), prevent coronary heart diseases (Howard, et al., 1996 and Morris, 1994), scavenge free radicals and enhance immunity (Chew, et al., 1996). Beta-Cryptoxanthin and beta-carotene are also major sources of vitamin A (Wingerath, et al., 1995). Both zeaxanthin and lutein are reported to possess strong anti-tumor properties (Packer, et al., 1999). Epidemiological studies suggest that the antioxidant potential of dietary carotenoids may protect against the oxidative damage that can result in inflammation. A modest increase in dietary carotenoid intake is associated with a reduced risk of developing inflammatory disorders such as rheumatoid arthritis (Pattison, et al., 2005). Zeaxanthin and lutein cannot be synthesized by humans and animals, so these carotenoids must be obtained through the ingestion of food and/or dietary supplements.
Age-related Macular Degeneration (AMD) is the leading cause of blindness for people older than 65 in the United States, and is expected to affect 40 million U.S. residents by the year 2030 (Abel, 2004). Treatments to ameliorate the effects of the disease and methods for preventing the onset of the disease are desperately needed. Since lutein and zeaxanthin play a critical role in the protection of the macula, it is important that people have access to these compounds, either through dietary sources, through supplements, or through so-called functional foods that contain enhanced levels of these nutrients. Numerous epidemiological studies suggest that the typical intake of lutein and zeaxanthin is only in the 1-3 mg/day range, see Brown et al. (1999) and Lyle et al. (1999). Seddon et al. (1994) reported a relationship between the intake of lutein and zeaxanthin at 6 mg per day and a decreased risk of AMD and cataracts. This dietary gap of 3-5 mg per day can be eliminated with the use of supplements.
A higher dietary intake of carotenoids is also associated with a lower risk for AMD (Age-related Macular Degeneration) occurring in older adults. Hereditary forms with an early onset include Stargardts, Best's Disease and progressive Cone Dystrophy. Hereditary retinal degenerations that attack the whole of the retina tend to be more severe. The most common types of these diseases are Retinitis Pigmentosa, Choroideremia, Ushers Syndrome and diabetic retinopathy. Individuals consuming the highest levels of carotenoids exhibit a 43% (statistically significant) lower risk for AMD (Seddon, et al., 1994). The specific carotenoids, zeaxanthin and lutein, are most strongly associated with a reduced risk for AMD. Zeaxanthin and lutein are the sole xanthophyll pigments found in the retina and concentrated in the macula. Excellent reviews of the role of carotenoids in the macula are found in Davies, et al., (2004), Stahl, et al., (2005), Stringham, et al., (2005), Ahmed, et al., (2005), Stahl, (2005), Beatty, et al., (2004), Davies, (2004) and Alves-Rodrigues, (2004).
There is a strong association between higher consumption of dark green vegetables, which contain xanthophylls, including zeaxanthin and lutein, and a decreased risk for light-induced oxidative eye damage, such as cataract formation, see Brown, et al. (1999) and Ribaya-Mercado, (2004). Although dark green vegetables are an excellent dietary source of zeaxanthin and lutein, the isolation and purification of these compounds in large quantities from green vegetables is time-consuming and costly. Twenty-five grams of a fresh, dark green vegetable such as kale theoretically provide 10 mg of lutein (Khachik, et al., 1995). Corn, one of the highest plant sources of zeaxanthin, contains about 0.528 mg of zeaxanthin per 100 grams of corn (Lutein and Zeaxanthin Scientific Review, Roche Vitamins Technical Publication HHN-138210800). It would require 1.9 kg of corn or 0.623 kg of peppers to provide 10 mg of zeaxanthin from these sources.
Therefore, a highly concentrated source of natural zeaxanthin is needed for the manufacture of dietary supplements and functional foods. Moreover, zeaxanthin is an important ingredient to add color to foods and as an additive in animal feeds to color poultry skin, egg yolks, fish flesh and the like. A natural source of zeaxanthin that can be used in foods is preferred and/or regulated over a synthetic product in these applications.
Cis-trans Forms and Stereoisomers
Zeaxanthin from natural sources is generally obtained in the form of an all-trans isomer. It is well known that the trans isomer can be converted to cis forms by the application of heat and/or light or by the addition of a catalytic amount of iodine [(Zechmeister, (1962); Khachik, et al., (1992); Updike, et al., (2003); Englert, et al. (1991) and references therein; Karrer and Jucker, (1950)]. Zechmeister also discusses isomerization by acid catalysts, contact with active surfaces, via boron trifluoride complexes and biostereoisomerization. Given the number of double bonds in the structure, a large number of different cis isomers are possible. Both cis and trans isomers have been detected in the human retina, but it is thought that the all-trans form is the desired isomer for providing protection to eye tissues. An excellent review of cis-trans isomerization of carotenoids is given by Schieber and Carle (2005).
Zeaxanthin also exists in two enantiomeric and one meso form, namely 3R,3′R; 3S,3′5 and 3R,3′S (note 3S,3′R is identical to 3R,3′S). All three stereoisomers have been found in the human retina (Howard, et al., U.S. Pat. No. 6,329,432), but the 3R,3′R isomer is dominant. It is difficult to separate these three isomers of zeaxanthin from each other in commercial quantities for human consumption. Therefore, for synthetic production of zeaxanthin, either a chiral process or a chiral separation process is needed in order to purify and produce the 3R, 3′R stereoisomer.
Zeaxanthin Needs in the Marketplace
There is a perceived need in the marketplace for naturally derived zeaxanthin, as opposed to synthetic zeaxanthin, that can serve as a dietary source in the form of a dietary supplement, a food or beverage additive, or a food or beverage colorant. Furthermore, there is a need for zeaxanthin for dietary supplements, food or beverage additives, and food or beverage colorants in biologically available forms.
There is also a need for naturally derived zeaxanthin, as opposed to synthetic zeaxanthin, that can serve as an additive in animal feeds, such as poultry feed, to color flesh and skin, egg yolks and fish flesh. Certain types of poultry feed additives prepared from corn gluten contain a relatively high percentage of zeaxanthin (about 15-30%), when measured as a percentage of total carotenoids. However, the total carotenoid content of these feed additives is very low (only about 100 milligrams of total carotenoids per pound of poultry feed). Another type of poultry feed additive is prepared from marigold extracts. This additive contains roughly 100-200 times as much yellow pigment per pound of additive (i.e., about 10 to 20 grams of lutein and zeaxanthin per pound); however, more than 95% of the yellow pigment in this marigold preparation is lutein, not zeaxanthin. Zeaxanthin comprises only about 2 to 5% of the yellow pigment in this poultry feed additive (Garnett, et al., U.S. Pat. No. RE 38,009).
Plant Sources of Zeaxanthin
The public generally prefers to consume compounds that are derived from natural sources as opposed to those that are produced synthetically. Natural sources containing high levels of zeaxanthin currently include certain mutant varieties of marigold flower petals, berries of the genus Lycium and Physalis, and specifically Chinese wolfberries (Lycium chinense). Preferred materials containing zeaxanthin include fruits like oranges, peaches, papayas, prunes, and mangos (Levy, U.S. Pat. No. 6,191,293). Todd, et al., (U.S. Application Publication No. 2006/0185034) describe a Capsicum plant that expresses very high levels of zeaxanthin; hereafter known as “orange paprika” or the oleoresin derived from it as, “Todd et al. Capsicum oleoresin”. Todd, et al. describe the dried ripe fruit pod flesh as containing greater than 0.4% zeaxanthin, as measured as the free-form of zeaxanthin, and exhibiting a percentage of zeaxanthin relative to total carotenoids of greater than 50%.
While the present invention is directed in particular to the saponification of zeaxanthin esters extracted from Capsicum and isolation of free-form zeaxanthin in high yield and state of purity, one skilled in the art would be able to apply these teachings to zeaxanthin from other sources and to other xanthophyll derivatives obtained from a variety of sources. Other xanthophylls which may be saponified/isolated according to the present invention include lutein, beta-cryptoxanthin, capsanthin, capsorubin, antheraxanthin, violaxanthin, and the like.
Beta-carotene and other carotenoids are desirable materials to isolate for nutraceutical applications since it is reported they quench active oxygen species such as singlet oxygen without damage to themselves. They can do this repeatedly, converting singlet oxygen back to ground state oxygen, thereby preventing singlet oxygen from causing damage, which damage may lead to cancer, including skin or lung cancer. Beta-carotene is also a very efficient free-radical trap (Biyani, et al., 2000). The methods for saponifying and isolating xanthophyll esters of the present invention may be used to isolate carotenes which may be present with the xanthophylls in a purified form.
Saponification
Zeaxanthin from natural sources usually exists as a mixture of free xanthophyll compounds together with the pigment in the form of mixtures of mono and diesters of fatty acids. The fatty acids generally contain from eight to twenty carbon atoms. Zeaxanthin of Capsicum, typically is a mixture of these three forms in combination with fatty acids such as include linoleic, palmitic, oleic, linolenic, myristic, stearic, lauric, and the like.
Many methods for converting these esterified forms of zeaxanthin to their free alcohol forms are known and documented. Methods for preparing esters from the non-esterified form are also known and documented. Saponification is the conversion of the fatty acid ester into alcohols and the alkali salts of the fatty acids (soaps) by treatment with a base such as sodium or potassium hydroxide. After saponification, xanthophylls are often further purified by recrystallization and/or chromatographic techniques.
Zelkha, et al., (U.S. Pat. No. 6,797,303) teach a process for extracting plant matter. Zelkha, et al. teach that one should first wash plant matter, such as Chinese wolfberries, with water to lower the Brix of the aqueous phase to less than or equal to 10° Brix, before milling and separation of the remaining plant matter (pulp) by decanting or centrifuging, and prior to extraction of the pulp with an organic solvent to create an oleoresin. Zelkha, et al. teach that when unwashed plant matter having a Brix greater than 10° is extracted, the subsequent separation of the pulp from the extracting solvent is problematic due to generation of three phases which are difficult to separate, when the plant material before extraction is not dried, and the resulting oleoresin is of poor quality, low content of the desired lipophilic substance, i.e. carotenoids and is unsuitable for use for further isolation of the carotenoid contained therein.
Zelkha, et al., report a process for extracting carotenoids from plant matter, whose Brix is greater than 10° and making an oleoresin from that extract. Their process focuses on getting the Brix below 10° prior to solvent extraction. During this process, they saponify the oleoresin derived from washed wolfberry plant material having less than 10° Brix, to liberate the free form of the zeaxanthin. The saponification reaction of Zelkha, et al., is carried out at a temperature of 70° C. to 80° C. in a mixture containing an aqueous solution of potassium hydroxide, ethanol and hexane for about 1 hour. Upon hydrolysis of the zeaxanthin diester-containing oleoresin, zeaxanthin crystals precipitated and the mixture was filtered. The solid fraction contained about 70-90% zeaxanthin, but no yield was given. No further detail was given, no further purification was described.
The process of the present invention starts with oleoresin that has not been subjected to the pre-extraction procedures taught by Zelkha, et al. and while it also uses aqueous potassium hydroxide, an alcohol and a low boiling hydrocarbon, there are other important differences. First, in the instant process the starting material is derived from paprika pods and not Chinese wolfberries. Further, the instant process is suitable for saponifying oleoresins that contain relatively low levels of xanthophyll esters. The Zelkha et al. process does not contain sufficient purification steps to achieve high levels of purity from these kinds of oleoresins. Second, the zeaxanthin yields (up to 80%) and purity (up to 70%) in the instant process are very sensitive to the workup conditions, and it is these workup processes that are the subject of this disclosure. Reactions 24 through 27 in Example 1 of the instant invention give representative yields and purities that were obtained by running a similar saponification procedure to that described by Zelkha, et al. on Capsicum oleoresin. Yields ranged from 45% to 65% with purities ranging from 9.2% to 20.1%. It should be noted that the 20.1% purity material was obtained in only 45% yield.
Ausich, et al., (U.S. Pat. No. 5,648,564) describe a method for producing xanthophyll crystals using propylene glycol and aqueous alkali, which crystals are free of trapped solvents. Ausich, et al. teach a method of forming xanthophyll crystals without the use of relatively toxic organic solvents during isolation or crystallization. Furthermore, Ausich, et al. teach that wolfberries (Lycium barbarum) are an excellent source of zeaxanthin, and use solvent free zeaxanthin oleoresin derived from wolfberries as starting material for saponification and crystallization using propylene glycol and aqueous alkali. No yield or purity of zeaxanthin is reported in the example for the extraction, isolation and purification of zeaxanthin from wolfberries. Ausich, et al. report that for lutein oleoresin derived from marigold flowers (Tagetes erecta), 59 percent of the total carotenoids were recovered from the starting oleoresin, and that 93.1 percent of the carotenoids were lutein. Ausich, et al. mention that similar manipulations using an oleoresin from dried red peppers of Capsicum annuum in place of the ground marigold flower petal oleoresin provides a mixture of capsanthin and capsorubin crystals; however, no data regarding yield or purity of capsanthin and capsorubin is provided.
For purposes of comparison, the (Ausich, et al.) process was performed using zeaxanthin oleoresin obtained from Capsicum extraction, and although the reaction went to completion (>99% HPLC yield of free form zeaxanthin), the isolated free-form zeaxanthin was obtained in only 54.6% yield and 31.6% purity.
Grant, et al., (U.S. Pat. No. 3,523,138) teach a method of making a xanthophyll product from marigold flowers by contacting marigold petals or an oily extract of marigold petals with an alkali in water and with alcohol at reflux for up to 24 hours to saponify the lutein diesters, thus liberating free lutein. The resulting lutein was separated by extraction from the reaction mixture with an organic solvent such as isopropyl ether, chloroform or ether. The organic solution was then evaporated to obtain a solid containing the free lutein.
These workers did not use the isolation procedures described in the instant invention. Their source of xanthophylls is limited to marigold only, and it has been shown that the source of xanthophylls is important with respect to how the material will respond to a given saponification and xanthophyll isolation procedure. They further do the saponification in water, alkali and an alcohol such as methanol or ethanol. They do not use a hydrocarbon in the saponification reaction itself, which has been shown to be advantageous in the process of the present invention. They also teach neutralization of the saponification reaction mixture, which has been shown to be problematic for xanthophyll recovery and purity in the current invention. Grant, et al., also teach extraction of lutein with an ether or chloroform, which solvents are not used in the process of the present invention.
Khachik, (U.S. Pat. No. 5,382,714) describes a method for the isolation, purification and recrystallization of lutein starting with saponified marigold oleoresin, which contains free lutein. The saponified marigold oleoresin was prepared by treating an organic solvent extraction of dried marigold flowers (Tagetes erecta) with an alkaline solution at 65° C. until greater than 98% of the lutein existed in the free form. The product was then homogenized with distilled water and ethanol (2.3:1 volume ratio) at room temperature for 30 minutes. The mixture was filtered and the filtrate discarded. The isolated orange precipitate was washed with distilled water until the filtrate was nearly colorless and the pH was neutral. The precipitate was then washed sequentially with cold (0° C. to 5° C.) ethanol and hexane, respectively. The resulting lutein was 70% pure by spectroscopic analysis, but no yield was reported or is calculable. Final purification was accomplished by recrystallization from a 1:1 mixture of dichloromethane and n-hexane. The solution was kept cold −20° C. to −10° C. for 3 hours. The resulting crystals were then filtered and washed with cold n-hexane (0° C.) and dried under vacuum. The purity was greater than 97%. A yield is not reported for this process. While high purity is important, to run a commercially viable and successful process, one must have high yield as well.
When a similar process was used for the Todd et al. Capsicum oleoresin, such as reactions #1-13 in Example 1, lower purities and yields were obtained. The highest purity obtained was 31.7% and the corresponding yield was 45%. Hexane was needed during the saponification to obtain higher yields and purities. Additionally, it was shown with the process of the present invention, that only two water washes could be done, because a third wash resulted in a persistent emulsion, which made further workup extremely difficult. The process described in the instant invention provides yields of from 60 to 80%, of materials suitable in purity for use in beadlet manufacture and nearly quantitative recovery of all input xanthophylls, whose fractions have differing states of purity. The reaction yields for liberation of zeaxanthin from its esterified form is typically >99% and isolated yields of free-form zeaxanthin from the saponification reaction are about 60 to 80% corresponding to >99% of input all-trans-zeaxanthin. An additional 10 to 20% of zeaxanthin is recoverable by precipitation on standing from the methanol supernatant with a purity of 2 to 20%. The remaining 10 to 20% yield is recoverable from the methanol supernatant by desolventization, but its purity is only about 1 to 2% and it contains an abundance of cis-isomers.
Khachik, (U.S. Pat. No. 6,262,284) describes a process for liberating zeaxanthin from its esterified form from Chinese wolfberries using tetrahydrofuran (THF), an alcohol and 5% or 10% potassium or sodium hydroxide, maintained at pH=12 for 1 to 2 hours at room temperature. In the first example of Khachik, the free zeaxanthin was isolated by filtering the reaction mixture and it is stated that the solids formed were washed with tetrahydrofuran. The solvents were evaporated under reduced pressure and the solids were stirred with water and ethanol. The mixture was then centrifuged and the solids washed two more times so that the pH of the aqueous wash was pH 7. The solids were then washed with ethanol, centrifuged and dried under vacuum to give orange crystals containing 75% zeaxanthin. Silica gel treatment was also listed as an option for increasing the purity of the 75% zeaxanthin to 97%. Lastly, the zeaxanthin could be recrystallized from THF and water to provide zeaxanthin in purities up to 97% or greater. One drawback of the Khachik process is that it uses tetrahydrofuran, which is an ether that readily forms peroxides. Although Khachik states that THF is “quite safe for commercial production”, it can present a dangerous explosion hazard on a production scale. Another drawback of the Khachik process is the expense associated with the use of a silica gel process step. In addition, since no yields were reported or calculable from the data presented, it is difficult to gauge the industrial feasibility of the Khachik process.
Kimura, et al. (1990) report on the saponification of synthetic carotenoids and natural carotenoids in food samples (tomato, kale, papaya) using different procedures. They report that conflicting results exist in the literature on the stability of carotenoids to saponification and the purpose of their study was to resolve those conflicts. Kimura, et al. reported that hot saponification resulted in greater losses of carotenoids, together with the formation of cis isomers and epoxycarotenoid byproducts as well. Kimura, et al. also note that degradation of carotenoids was further aggravated by contact between the carotenoids and the alkali.
Kimura, et al. teach that using petroleum ether makes the saponification conditions less harsh due to temperature reduction and a protective effect on the released carotenoids. They also reported that carotenes were more stable than xanthophylls to saponification. Running the saponification in alcohol vs. an alcohol and alkane mixture gave higher percentage losses of beta-carotene. However, the conditions such as alcohol type (methanol vs. ethanol), base concentration (10% vs. 60%), and times were not identical in the comparison of the with- and without-petroleum ether cases. This makes a direct comparison difficult.
We have not observed this phenomenon and have stability data that demonstrates that zeaxanthin is stable in aqueous methanolic potassium hydroxide for at least 15 days with no loss in zeaxanthin content or decrease in trans:cis ratios (see Example 2). Kimura, et al. further report that saponification of carotenoids at room temperature in petroleum ether for 16 hr, with an equal volume of 10% methanolic potassium hydroxide was the preferred method for retaining beta-carotene, gamma-carotene, beta-apo-8-carotenal and lycopene; however, even under these mild conditions, lutein, zeaxanthin and violaxanthin from kale degraded significantly. Kimura, et al. noted that these losses could be reduced to insignificant levels by using an atmosphere of nitrogen or an antioxidant. Kimura, et al. report the levels of zeaxanthin as means and standard deviations of duplicate determinations using this method of cold overnight saponification of the zeaxanthin composition of kale, as shown in Table 1.
TABLE 1Levels of zeaxanthin (ug/g) in kale.SaponifiedSaponifiedunderwithCarotenoidUnsaponifiedSaponifiedNitrogenpyrogallolZeaxanthin4.4 +/− 0.7*1.8 +/− 0.1**2.2 +/− 0.3*1.9 +/− 0.0*
The values in the row having the same superscript were reported as not significantly different (p≦0.05). It is not stated, but presumed that the concentrations reported are calculated back to the concentration that would have been in the plant material. However, for duplicate measurements, the confidence intervals are extremely large, and this “significant” difference can be misleading. If these means and the standard deviations remained the same for more replicates, they would be considered significantly different by Tukey's test. It appears that on an absolute basis, that there is very little difference between the saponified, the saponified under nitrogen and the saponified with pyrogallol results within one standard deviation. There appears to be about a 50% loss between the unsaponified material and the saponified materials in general.
Using the method of the present invention and Capsicum starting material, a completely different result is obtained. We observe <1% loss of zeaxanthin and furthermore we observed an increase in the amount of the trans isomer to that of cis isomers.
Granado, et al. (2001) describe the comparison of two analytical procedures for the preparation of xanthophyll samples for HPLC and UV-VIS analysis. Granado, et al. describe saponification reactions in ethanol with pyrogallol, 40% potassium hydroxide under nitrogen and in the dark. The workups include partitioning between water and dichloromethane/hexane in one case vs. water and diethyl ether/petroleum ether. The two methods were found to give equivalent analytical results. Granado, et al. do not teach a process suitable for isolating free zeaxanthin on a commercial scale, rather Granado, et al. teach a sample preparation for an analytical method.
Montoya-Olvera, et al., (U.S. Pat. No. 6,504,067) describe an industrial process to obtain xanthophyll concentrates of high purity from plant extracts comprising: refining the plant extracts by treating them with a diluted alkali, followed by treating them with a diluted organic acid or inorganic acid in order to eliminate impurities and obtain a refined extract. The refined extract was saponified by means of a strong alkali aqueous solution at 90° C. under nitrogen, treating the saponified mass with a dilute organic or inorganic acid, followed by several water rinses to a neutral pH in order to separate a xanthophyll concentrate and finally removing any remaining impurities by extracting with hexane. These final steps produce a concentrate that is >95% xanthophylls with 94.5% recovery of the original xanthophylls.
Montoya-Olvera, et al., teach that the acidity, or acid value, of the input oleoresin and the acid:alkali ratio used in the pretreatment step is a key parameter for the process. However, Montoya-Olvera, et al. do not teach how to determine the acid value, and accepted methods (such as AOAC, ASTA, Food Chemical Codex) for determining acidity of an oil are based on titration using a phenolphthalein indicator, which is extremely difficult to visualize in solution with a dark red or orange oleoresin. Montoya-Olvera, et al. teach that capsanthin and capsorubin concentrates may be obtained when Capsicum is used as the starting material, although no specific examples are provided, which examples may have demonstrated how the acid value may be measured in a dark red or orange oleoresin; moreover, no data regarding yield and purity is given. Montoya-Olvera, et al. teach that lutein and zeaxanthin concentrates are obtained when Tagetes oleoresin is used as the starting material and provide examples for the isolation of such concentrates which are derived specifically from Tagetes plant material.
When applied to Todd et al. Capsicum oleoresin, this process fails at the very first step, in that we were unable to achieve a separation even after centrifuging for 6 hours (See Example 10). Even if it was possible to carry out the process on Capsicum oleoresin, the Montoya-Olvera, et al. process is clearly different from that described in the present application in that we use lower temperatures and a hydrocarbon solvent in our reaction to help improve the purities and yields of our final product.
Bhaskaran, et al., (U.S. Pat. No. 7,179,930) teach a process to saponify xanthophylls using phase transfer catalysts in an alcoholic medium. Examples of these phase transfer catalysts are quaternary phosphonium salts and quaternary ammonium salts.
Madhavi, et al., (U.S. Pat. No. 6,380,442) teach a process to isolate carotenoids, especially lutein, from a lutein source, such as marigold oleoresin, using isopropyl alcohol, water and alkali, for a minimum of 60 to 90 minutes at a temperature of about 60° C. to 65° C. The hydrolyzed carotenoids were precipitated from the reaction mixture by addition of water and the precipitate was recovered by centrifugation, followed by repeated water washings and a drying step to provide a fine crystalline material. Madhavi, et al. teach a process for isolating carotenoids from marigold oleoresin, the preferred starting material, with minimal use of organic solvents. The process is not attractive for commercial applications because the water required is more than 30 times per kilogram the input material.
A similar process was performed using Capsicum oleoresin containing 2.7% zeaxanthin present mainly in its esterified form, using 45% potassium hydroxide in isopropyl alcohol, wherein heptane was also added to the reaction mixture. The resulting free-form zeaxanthin product had a purity of 2.9%, with a corresponding isolated zeaxanthin yield of 48% (See Example 23). Typically adding a hydrocarbon solvent with an alcohol provided higher yields and purities than the case where no hydrocarbon was added with the Todd et al. Capsicum (See Example 1).
Kumar, et al., (U.S. Pat. No. 6,743,953) describe a process for the isolation of high purity xanthophyll crystals (at least 85% total xanthophylls, and at least 90% trans lutein and/or zeaxanthin) which comprises admixing and heating xanthophyll ester concentrate with excess alcoholic alkali solution, maintaining the resulting mixture at a temperature in the range of 65° C. to about 80° C., for a period sufficient to saponify the xanthophyll esters, removing the aliphatic alcohol from the mixture under reduced pressure to obtain the crude saponified concentrate. The crude mixture was then admixed with water at room temperature to form an oily mixture, which was extracted with ethyl acetate three times to produce a xanthophyll extract, which was washed two times with water. The ethyl acetate was distilled off under reduced pressure to recover ethyl acetate and to produce the xanthophyll concentrate. The concentrate was then admixed with a solvent or mixture of solvents, preferably acetone and hexane at room temperature with stirring. The xanthophylls separated out as crude crystals and were removed by filtration. Finally the crude crystals were further purified by washing with an aliphatic alcohol and vacuum dried to obtain xanthophyll crystals that were at least 85% xanthophylls by weight and greater than 90% trans lutein by HPLC. The disadvantages of the Kumar, et al. process are the low isolated yields (see Table 2), multiple distillation steps, the multiple washing steps, and the crystallizing, filtering and vacuum drying steps. These are costly steps in a commercial process and make the process inefficient and unlikely to be cost effective.
TABLE 2Reported inputs and outputs and yields thereby calculatedfrom Kumar, et al., (U.S. Pat. No. 6,743,953).reported inputsreported outputsexam-% xantho-% xantho-calculatedple#massphyllsmassphyllsyield157.9811.54%1.9886.23%26%256.311.82%1.9388.69%26%351.611.82%2.1190.07%31%45011.82%2.1190.21%32%547.311.82%1.5291.34%25%650.411.82%2.29989.05%34%751.611.82%1.99685.73%28%847.311.82%1.7381.41%25%95011.82%2.7890.58%43%30%<-- average
In addition, the relatively low solubility of zeaxanthin in ethyl acetate (ca. 1.07 gram/liter; see Example 11) equates to a relatively small amount of oleoresin that can be converted to free-form zeaxanthin in any given vessel, when compared to the process described in the instant invention. For example, it would require more than 1000 gallons of ethyl acetate to dissolve 10 pounds of zeaxanthin whereas in the instant invention (see Example 6), approximately 10 pounds of zeaxanthin, present in the input oleoresin predominately in esterified form, were saponified in a 400 gallon vessel. To run the same amount of oleoresin using the process described in Kumar, et al. would require a much larger vessel and which may limit the use of the Kumar process for industrial scale purification of zeaxanthin.
Tyczkowski and Hamilton (1990) describe the extraction of free form lutein from a commercial source of saponified lutein and the further crystallization of that extract to produce analytically pure standards. The method of Tyczkowski and Hamilton uses a combination of four solvents in the extraction process (hexane:acetone:toluene:absolute ethanol in a 10:7:7:6 vol/vol/vol/vol ratio). Toluene is a known teratogen and ethanol is a controlled substance. These two factors alone make this an impractical method for a commercial process to purify lutein. Recycling of such a complex solvent mixture on a large production scale also presents costly challenges. The procedure further involves low temperature (4° C.) crystallization to produce the final product. Low temperature crystallization is also a costly procedure.
Sas, et al., (U.S. Pat. No. 5,876,782) teach a method of treating natural plant material in situ using alcohol and a base capable of transesterifying the esters of xanthophylls such that free form xanthophylls were formed as well as the esters of the fatty acids. The reaction was neutralized with acid and solvent removed by drying. The resulting solid was then isolated as the product. Sas, et al. further describe that the process does not involve the use of an aqueous solution, but instead, use an alcohol in situ. Sas, et al. teach the use of neutralization with acid, which has been shown to be problematic in the process of the present invention. Additionally, the Sas, et al. process has no additional steps, such as washing or partitioning, to increase purity and purities were not reported for their isolated products.
Transesterification forms esters of the fatty acids, rather than the salts of the fatty acids. This is a different process than saponification, and as such would imply differences in the workup and isolation of product. Sas, et al. even state that their invention “does not require the organic extraction step, nor the saponification step, to provide an improved plant material.” Thus, Sas, et al. recognized that the processes are different.
Sas, et al. provide an example for the liberation of capsanthin from red paprika. No yields were reported. Since the subject of the Sas, et al. disclosure is the conversion of esterified to free-form xanthophylls in situ in plant material, the concept of ‘purity’ is not addressed.
Sas, et al. do not describe the method of the present invention; the highest purity of zeaxanthin achieved from transesterification reactions using methanol and sodium methoxide was 45.8%, with a yield of 58% (See Example 1).
Sas, et al., (U.S. Pat. No. 6,221,417) describe a human food colorant composition and a process obtaining such food colorant composition. Sas, et al. convert the esterified forms of xanthophylls to free-form xanthophylls via transesterification using a base such as potassium hydroxide at pH=11 to 14, in dry methanol for 10 hrs at 69° C. The resulting mixture was neutralized with phosphoric acid after which the solvent was removed via distillation over 16 hours at 69° C. The residue was dried at room temperature or via vacuum drying at a temperature less than 50° C. Sas, et al. provide an example for the liberation of capsanthin from red paprika. No yields are reported. Since the subject of the Sas, et al. disclosure is the conversion of esterified to free-form xanthophylls in situ in plant material for a food colorant, the concept of ‘purity’ is not addressed. An indication of the range of purities obtainable by this procedure is summarized in Sas, et al., “a fine powder with a xanthophyll activity of 10-14 g/kg is obtained”. The fine powder isolated via the process described in the instant invention has at least 50 times greater ‘xanthophyll activity’.
Rodriguez-Bernaldo de Quiros and Costa (2006) analyze and review carotenoid compositions found in vegetable material and human plasma. Rodriguez-Bernaldo de Quiros and Costa discuss issues with saponification of xanthophylls and provide several references indicating that xanthophyll recovery is very sensitive to temperature and reaction conditions.
Khachik, et al., (1986) report an important loss in the xanthophyll content of raw broccoli after applying a treatment of 30% methanolic potassium hydroxide under nitrogen atmosphere during 3 hr, however the loss of carotenes was not significant. This understanding in the art with regard to xanthophyll loss related to saponification is supported by the teaching of Granado, et al., (1992) who also report a loss in the concentration of xanthophylls related to saponification. Furthermore, Scott, et al., (1992) state that “Depending on the nature of the carotenoid, saponification may result in destruction or structural modification.”
This makes the resulting reaction yields from the process of the present invention all the more surprising. There is no significant loss in xanthophylls using 45% potassium hydroxide under reflux for up to 40 hr (see Example 3) nor was any significant loss of zeaxanthin observed after allowing the mixture to sit in strongly basic (pH˜14) solution at room temperature for 15 days (Example 2). In fact, we further see an increase of the more highly-desired trans isomer via isomerization of the cis isomer of zeaxanthin under these conditions.
Hart and Scott, (1995) employed mild saponification conditions to fruits and certain vegetables, like peppers, in a process using methanolic potassium hydroxide (10%) under nitrogen, in the dark for 1 hr at room temperature. Hart and Scott found that these mild conditions gave the maximum values for the carotenoids of interest. Hart and Scott further teach that for higher fat content materials, a higher concentration of potassium hydroxide is required.
Larsen and Christensen (2005) describe an analytical method for carotenoid analysis that uses a strongly basic resin to gently saponify the chlorophyll and carotenoids present in leafy green vegetables. This saponification involves the use of an acetone extract of the leafy material followed by treatment with base for 30 minutes followed by filtration. The initial concentration of the carotenoids in the samples submitted to saponification were between about 0.0002% to 0.0023%, which carotenoids were further reduced in concentration during the saponification step. This is fine for an analytical procedure, but a very low concentration for a commercial process. The concentrations achieved in the process of the present invention are about 200 to 2000 times greater than the Larsen and Christensen procedure. Larsen and Christensen discuss the method of Khachik, et al., (1986) and observe similar recovery percentages for standard treatment with methanolic potassium hydroxide, and which recovery percentages are lower for all carotenoids, except beta-carotene. Larsen and Christensen attribute the poorer recovery in the Khachik, et al. procedure to longer contact times with hydroxide ions resulting in oxidation and isomerization due to the many laboratory operations involving multiple extractions and evaporations.
Majeed and Murray, (WO 02/060865) teach a process for saponifying and isolating a composite mixture of zeaxanthin, cryptoxanthin, capsanthin, capsorubin and/or beta-carotene from a special variety of chili (Capsicum) known as the Byadagi/Chappatta. The process involves treating an acetone-extract oleoresin made from the Byadagi/Chappatta chili with ethanol and aqueous alkali for 2 to 8 hours at room temperature. The reaction mixture was diluted with 4 parts of a mixture of alcohol and water in a 1:1 ratio. The mixture was extracted with ethyl acetate or toluene or a mixture of the two. The organic layer was washed with water to remove the alkali, and then concentrated to one-third to one-quarter the original volume to initiate crystallization of the zeaxanthin. The crystallization was kept at 0° C. to 5° C. to complete the crystallization, after which it was filtered and dried at 35° C. to 75° C. with high vacuum for 10-48 hours. Majeed and Murray describe using dichloromethane in place of the ethyl acetate and/or toluene extraction solvent above.
We obtained samples of Byadagi/Chappatta chilies (the latter designated S-66) from India, analyzed them for their zeaxanthin content, and compared the results with those of the inventive Capsicum cultivar described in Todd et al. (U.S. Application Publication No. 2006/0185034). It should be noted that ASTA values are used as a proxies for molar or weight ratios of pigments. ASIA values are calculated from the absorbance of a solution of the extract at a wavelength of 460 nm. The Todd, et al. Capsicum (orange paprika) extract have a maximum absorbance at 454-455 nm. Evidence that the samples of the chilies we obtained were similar to those described by Majeed and Murray comes from the similar levels of color reported as ASTA color values (See Example 23 of Todd, et al. (U.S. Application Publication No. 2006/0185034 for method and calculations). The Chappatta chili in Majeed and Murray is reported to have an ASIA value of 125.26. The Chappatta chili we analyzed had an ASIA color value of 125.5. The Byadagi chili in Majeed and Murray is reported to have an ASTA color value of 156.9, while the Byadagi chili we analyzed had an ASIA color value of 213.6. The higher color value in the Byadagi chili we analyzed indicates a higher carotenoid pigment level. If one assumed that all of the absorbance was due to zeaxanthin, one would over-estimate the actual amount of zeaxanthin in the chilies Majeed and Murray used. Analysis of the Byadagi chili gave a total zeaxanthin content of 0.0325% of the dry fruit weight. The percent zeaxanthin to total carotenoids was measured as 6.23%. Analysis of the Chappatta chili gave a total zeaxanthin content of 0.0313% of the dry fruit weight. The percent zeaxanthin to total carotenoids was measured as 7.10%. The weight percent of zeaxanthin in the dried pods of the Byadagi and Chappatta chilies are nowhere near as high as the levels of the Todd et al. Capsicum. Likewise, the percent zeaxanthin to total carotenoids values for the Byadagi and Chappatta chilies do not approach the levels of the Todd et al. Capsicum. Since only very low levels of zeaxanthin are present in the Byadagi/Chappatta chili, the solubility limitation of zeaxanthin in ethyl acetate (See Example 11) is not a concern in the process of Majeed and Murray. However, when larger amounts of zeaxanthin are being processed, very large volumes of ethyl acetate would be required on an industrial scale due to the relatively low solubility of zeaxanthin in ethyl acetate (about 1 gram/liter). Moreover, the low temperature crystallization of Majeed and Murray is not a cost-effective method for a commercial process of saponifying xanthophylls. A further disadvantage of this procedure is that toluene is a teratogen. Furthermore, ethanol is their preferred alcohol for saponification, but is a regulated substance and the measures taken to insure its' proper use make it costly and difficult to use in a commercial process in the U.S.
Pena, (U.S. Application Publication No. 2007/0161826) describes a process for saponifying oleoresin from marigold and boxthorn berries wherein the oleoresin is saponified with 50% aqueous potassium hydroxide at 103° C., followed by addition of an aqueous sodium chloride solution with stirring. The organic phase was then subjected to multiple sodium chloride and hexane washes, followed by the addition of methanol-water and phosphoric acid to the residual paste, then followed by a second methanol-water wash and subsequently three water washes and filtration. This was followed by another water-ethanol wash to provide a product which was 84.5% total carotenoids representing a yield of 68.7% from a marigold extract. This number of washes would be extremely costly on a large scale and sodium chloride would have a detrimental effect on stainless steel in plant equipment. Pena teach a similar procedure using boxthorn berries (Lycium chinensis) to provide a total yield of only 20.6% total carotenoids, 97% of which is zeaxanthin. Again, the cost effectiveness of the Pena process to obtain zeaxanthin is questionable, particularly given the recovery of the total carotenoids. Moreover, in the Pena process, many different solvents are used and these must either be recovered and purified to be used in other processes, or the mixed solvent streams for this process must be kept in their own tanks, putting a burden on inventory. Furthermore, ethanol is a regulated substance and the measures taken to insure its proper use are costly.
Xu, et al., (U.S. Application Publication No. 2007/0032683) describe a process for the isolation and purification of xanthophyll crystals from plant oleoresin using food grade solvents. The plant material Xu, et al. saponified included marigold flowers, kale, spinach, broccoli, corn, with marigolds being preferred. The Xu, et al. process comprises the steps of: saponifying the oleoresin in alcohol with aqueous alkali at a temperature of 40° C. to 85° C. for 3 to 5 hours, and then adjusting the pH to 1 to 7 after cooling. Two to ten volumes of water were then added, followed by 0.5 to 2 volumes of alcohol per weight of plant oleoresin. The temperature was then increased to 40° C. to 85° C. for 0.5 to 2.0 hours, during which time a crystalline precipitate formed. The crystals were recovered by centrifugation, then washed 2 to 3 times with water at 70° C. to 85° C. until the supernatant was colorless. The crystals were then leached with dry ethanol and dried in vacuum or freeze-dried. The Xu, et al. process may yield 80% total xanthophylls with at least 90% trans-lutein or zeaxanthin, depending on the plant material used as starting material.
The disadvantages of the Xu, et al. process using the Todd, et al. Capsicum is that attempts to decrease the pH from highly alkaline resulted in precipitation of large amounts of fatty acids and poorly compacted solids, resulting in much lower purities than the instant invention (See Example 9). Moreover, the Xu, et al. process specifically calls for the use of dry ethanol and, as mentioned previously, ethanol is a regulated substance and the measures taken to insure its proper use are much more costly than other solvents.
Sethuraman Swaminathan and Kunhiraman Priya Madavalappil (WO 2006/114794) describe a method for producing carotenoids rich in lutein from marigold flower petals. The process comprising the steps of: ensilage of marigolds, dehydration to obtain a dry meal, pelletization of the meal, solvent extraction of the pelletized meal with hexanes, hydrolysis of carotenoid esters with alkali after homogenizing the oleoresin in absolute alcohol, precipitation of the carotenoid crystals with a water and alcohol mixture, washing the precipitate with hot water to remove soaps and impurities, filtration of carotenoids, and drying to obtain crystals in high purity (>90% carotenoids having a minimum of 90% trans lutein) and high yield (55% to 80% isolated yields). The saponification and purification procedure involves homogenizing the oleoresin with absolute alcohol before adding aqueous alkali and heating at 70° C. to 80° C. for 30 minutes. This process is very different from the process of the present invention in that no alkane is used in the saponification process, furthermore, it is limited to marigolds as a source of carotenoids and we have shown that processes optimized for marigolds do not translate well to the Todd, et al. Capsicum (see Example 10 using the method of Montoya-Olvera, et al.). Moreover, the input marigold plant material is subjected to an anaerobic fermentation step to fix and enrich the carotenoids present in the marigold petals. This may provide a very different starting material than solvent extraction of the Todd, et al. Capsicum. A further disadvantage of this procedure for use with the Todd et al. Capsicum is that yields and purities of zeaxanthin are lower if the reaction is run only in alcohol without addition of an alkane (See Example 1).
Rosales, et al., (U.S. Pat. No. 7,150,890) describe the use of metallic halogenides, such as calcium chloride, magnesium chloride, etc. to precipitate fatty acids after xanthophyll oleoresins have been conventionally saponified with aqueous solutions of potassium hydroxide or sodium hydroxide between 80° C. and 120° C. for one to two hours, which provided a saponified marigold oleoresin containing about 30% xanthophylls, of which 94% was lutein and 6% was zeaxanthin. The precipitation was carried out at 35° C. to 50° C., followed by filtration and washing of the precipitate with a polar solvent such as an alcohol or acetone. The filtrates were combined and evaporated to a obtain a residue that contained 91 to 95% of the original amounts of xanthophylls in about the same proportions as the starting material. In only one of the examples do Rosales, et al. give an absolute purity obtained, which in that case was 90% total xanthophylls by weight. In the Rosales, et al. Process, zinc chloride (a Lewis acid), calcium chloride and magnesium chloride were used as titrants.
The disadvantages of this procedure for use with the Todd et al. Capsicum is that Rosales, et al. was working with marigold extract, and they precipitated solids from the supernatant along with lutein. They then extract the solids with acetone or alcohol to isolate the lutein. The purities of zeaxanthin were much lower when attempts were made to titrate the saponification mixture to a neutral or acidic pH (See Example 9) and purities were similar or lower by adding a chelator to try to remove metals, and a persistent emulsion occurred with the addition of calcium acetate (See Example 26).
Hoffman, et al., (U.S. Pat. No. 7,109,361) describe a process for the extraction of lutein and zeaxanthin from alfalfa. The overall yield of the process, reported in Example 1, was about 9%. The Hoffman, et al. process involved saponifying 27 lbs of oleoresin with 13 lbs of 40% aqueous potassium hydroxide at 140° F. for one hour. The pH was checked and adjusted if it was lower than pH 12. Next, 500 lbs of acetone was added to 40 lbs of saponified resin at 100° F., blended, and was subsequently separated into two phases via centrifugation. The acetone layer was evaporated to yield 10 lbs of an oil containing 6% lutein. The oil was mixed with 900 lbs of hexane at room temperature and cooled to −10° F. to induce crystallization. The hexane was removed and the crystals were rinsed with water in a 50:1 ratio, water to solids. The 0.6 lbs of crystals contained 75% lutein, which represented a 9% overall yield from the original amount of lutein in the alfalfa oleoresin. The disadvantages of the Hoffman, et al. process are the high volumes of solvent used for the small amount of lutein obtained and the low overall yield from the alfalfa. Additionally, the low temperature crystallization is a cost-inefficient process on a commercial scale.
Muralidhara, et al., (U.S. Pat. No. 5,602,286) describe a process for the recovery of xanthophylls from corn gluten. The Muralidhara, et al. process includes saponification and isolation steps. The saponification step is described to be a conventional saponification reaction. Muralidhara, et al., saponified with alcohol, specifically ethanol in the examples, and potassium hydroxide with the crude xanthophyll extract at reflux for one hour. The reaction mixture was then filtered and the filtrate was evaporated to obtain the refined xanthophylls (about 52% purity) in 47% yield.
The disadvantages of the Muralidhara, et al. process are that the relatively low yield and low purity of xanthophylls from corn. The reported yield is lower than that of the instant invention (60-80% isolated yields) and the purities were not as high as in the instant invention (50-70% purity). They also do not follow the steps of the process of the present invention. Furthermore, Muralidhara, et al. specifically uses ethanol as the alcohol used in the process. As mentioned previously, ethanol is a costly solvent to use in a commercial process.
Rodriguez, (U.S. Pat. No. 5,973,211) teaches a non-aqueous process for isomerization of lutein to epimers of zeaxanthin. The process includes a step for saponification of an oleoresin in the presence of a non-aqueous glycol, such as propylene glycol and/or glycerine, and sodium or potassium hydroxide with heating to 60° C.-80° C. for 50 minutes to 5 hours under nitrogen. The products of saponification were never isolated, since further steps were employed utilizing more base and higher temperatures to isomerize lutein to zeaxanthin.
Sanroma-Virgili, et al., (U.S. Pat. No. 5,998,678) describe a process for the preparation of free-form zeaxanthin via the saponification and isomerization of free-form lutein or lutein diesters or lutein derivatives where the hydroxyl groups are protected with lower alkyl, lower acyl, fatty acyl or a hydroxyl protector group, with an alkaline reagent (such as potassium hydroxide), a polar solvent or mixture from the group consisting of an ether, a polyhydroxyl alcohol, an ether-alcohol or a combination thereof, and wherein the lutein or lutein derivative is of natural or synthetic origin. The products of saponification were never isolated, since conditions were employed to go beyond saponification and to isomerize lutein to free-form zeaxanthin.
Torres-Cardona, et al. (U.S. Pat. No. 5,523,494) teach a process for the isomerization of lutein to zeaxanthin, which may include saponification of the plant extract material which is derived from marigolds or yellow corn. The process includes the slow addition of a strong alkaline base (such as potassium hydroxide) with a temperature from 25° C. to 180° C. for 5 to 96 hours, under an inert atmosphere to effect the conversion of lutein to free-form zeaxanthin. Although it is not specifically stated, the lutein sources listed would imply that an esterified lutein was the starting material. The intermediate, saponified, lutein was not isolated and characterized, and therefore, no yields are reported.
The Torres-Cardona, et al procedure is different from the method of the instant invention in that the important processing steps necessary to saponify and obtain high yields and purities of zeaxanthin from Capsicum extracts are not described.
Bernhard, et al., (U.S. Pat. No. 4,883,887) describe processes to make carotenoid structures synthetically. One sub-process describes using an alkali metal hydroxide or alkaline earth metal hydroxide in water, with an optionally inert solvent (e.g. ethers such as diethyl ether, diisopropyl ether, t-butyl methyl ether, tetrahydrofuran, dioxin and 1,2-dimethoxyethane, saturated and aromatic hydrocarbons such as hexane, cyclohexane, benzene and toluene, and the like) at temperatures from 0° C. to reflux. Bernhard, et al. use a synthetic source as starting material and do not disclose extraction/purification of carotenoids from plant material; a natural plant source is generally a more complex matrix, making separations and purifications more difficult. The ethers are dangerous solvents, in general, owing to their propensity to form explosive peroxides on standing. Great care must be taken when using these solvents industrially on a large scale. Additionally benzene is a known carcinogen and toluene is a teratogen.
Khachik, (U.S. Pat. No. 7,173,145) teaches a process for extracting and saponifying zeaxanthin esters from Chinese wolfberries utilizing tetrahydrofuran, an alcohol, and potassium hydroxide for one hour at room temperature. The solution was treated with aqueous hydrochloric acid to lower the pH to 7.0 to 7.5, after which the tetrahydrofuran and alcohol were distilled off under reduced pressure at 40° C. to 50° C. Hexanes were added and zeaxanthin was crystallized, centrifuged and vacuum dried at 60° C., resulting in a 81.7 yield (based on free-form zeaxanthin). This Khachik process uses ethers, which can be an explosion hazard due to peroxide formation and it also involves neutralization.
Attempts at neutralization immediately after saponification using the current invention on Capsicum extracts, resulted in a tremendous amount of precipitate (See Example 9), thereby complicating the isolation of zeaxanthin in high purities.
Minguez-Mosquera and Hornero-Mendez, (1993) describe an analytical method for identifying various carotenoid pigments in red peppers (Capsicum annuum). The analytical procedure requires the starting material be saponified. The Minguez-Mosquera and Hornero-Mendez saponifying procedure involves an ether extract solution being shaken with 20% potassium hydroxide in methanol solution for 1 hour. The aqueous phase was removed, and the organic phase was washed several times with water, dried over anhydrous sodium sulfate, and evaporated to dryness under vacuum at a temperature lower than 35° C. Minguez-Mosquera and Hornero-Mendez describe an analytical method for identifying individual carotenoid pigments, which analytical method does not pertain to large scale isolation/purification of carotenoids to obtain high yield and high purity.
This procedure differs from the process of the instant invention, in particular, ether is used as a solvent and the pigment composition in their Capsicum annuum red peppers is soluble in the organic phase. In contrast, zeaxanthin precipitates out of the aqueous/alcohol solution in the process of the present invention.
Processes for the saponification of paprika oleoresin to obtain the paprika pigments, the major pigments in the paprika being red pigments, and in particular, capsanthin, as the major carotenoid are described. Osamu, et al., (Japanese Patent Application No. 57-133160) describe the saponification of paprika oleoresin with alkaline water and/or alcohol followed by pH adjustment to acidic conditions with water to form two layers. The oil layer was collected and alcohol was added to it and the pigment was obtained by decantation of the liquid and dried. Osamu, et al., (Japanese Patent Application No. 57-180663) describe saponification of paprika oleoresin with the hydroxide, alcholate or carbonate of an alkali or alkaline earth compound, followed by addition of water, pH adjustment with acid to acidic conditions, and lastly extraction of the solids with an organic solvent such as acetone or ethanol and evaporation of the acetone to produce a paprika pigment. These two methods used pH adjustment to the acidic side, which may result in isolation problems, depending on the starting material, and lead to lower purities and yields of xanthophylls in the current invention. Masahiro, et al., (Japanese Patent Application No. 58-173164) describe a process where saponification was used as part of a larger process, the intent of the larger process is to deodorize paprika. Masahiro, et al. describe saponification of an oleoresin in aqueous solution, separating the solids and drying it prior to adding an organic solvent such as acetone or ethyl acetate to extract the pigment. The acetone extract was then concentrated and the concentrate steam distilled to provide deodorized paprika. Masahiro, et al. further describe that capsanthin was the major carotenoid in the oleoresin they were saponifying.
Curl, (1962 and 1964) describes saponification of red and green bell peppers for countercurrent distribution sample preparation. The peppers were blended with water and magnesium carbonate, then mixed with methanol. Celite 503 was added and the mixture was filtered.
Curl, (1953) describes a method for saponification that involves extracting oranges with diethyl ether and adding an equal volume of 20% potassium hydroxide in methanol to form a homogenous solution. The solution was allowed to stand overnight at room temperature, and was then diluted with water and extracted with ether. The ether layer was washed with water, dried over sodium sulfate and evaporated in vacuo.
The purification of xanthophylls from a saponified extract using a crystallization process is described in Rodriguez, et al. (U.S. Pat. No. 6,329,557). The process comprises the steps of dispersing the saponified extract in water to form a dispersion, mixing the dispersion under conditions such that a portion of any water-soluble compounds dissolves in the water to form an aqueous phase and a residue that is not soluble in water, adjusting the pH to between 1 and 7, preferably to between 5.0 and 6.5, then separating the aqueous phase from the residue, contacting the residue with a non-polar solvent under conditions such that a portion of any lipid-soluble compounds dissolves in the non-polar solvent and a portion of the xanthophylls precipitates from the non-polar solvent to form a precipitate, separating the non-polar solvent from the precipitate, washing the precipitate with a polar solvent such that at least a portion of any remaining chlorophylls dissolves in the polar solvent, and separating the polar solvent from the precipitate to yield a product comprising the xanthophylls at a desired level of purity.
This process differs from the instant process in two ways. The first is that it involves a pH adjustment to acid at a point where zeaxanthin is not soluble and a large volume of fatty acids would precipitate, making zeaxanthin isolation in high purity extremely difficult without further processing steps. The second is that it does not use hexane for the saponification reaction, but uses it for the workup. As will be shown later, one of the keys to achieving high purity zeaxanthin is the addition of an alkane to the saponification reaction at the beginning (see Example 1).
As can be seen from the foregoing analysis, there is a need for a process to convert xanthophyll esters derived from Capsicum sources to their free (non-esterified) forms in both high purity and high yield. The existing processes in the public domain, including those discussed above, fail to provide a process for Capsicum derived xanthophylls that meet the finished product needs for purity, yield, ease of use, industrial compatibility and cost that are required to make product suitable for the nutritional supplement, food and beverage industries. Moreover, the xanthophyll esters present almost exclusively in the trans form in the natural plant sources are often partially converted to their less valuable and less useful cis forms during extraction and further processing. The increased solubility of the cis forms leads to significant yield losses of xanthophylls, over all. Furthermore, there is a need for a process to re-convert cis product back to the more desired trans form.